Development of short‐target primers for species identification in biological studies of Carnivora

Abstract Noninvasive genetic sampling greatly facilitates studies on the genetics, ecology, and conservation of threatened species. Species identification is often a prerequisite for noninvasive sampling‐based biological studies. Due to the low quantity and quality of genomic DNA from noninvasive samples, high‐performance short‐target PCR primers are necessary for DNA barcoding applications. The order Carnivora is characterized by an elusive habit and threatened status. In this study, we developed three pairs of short‐target primers for identifying Carnivora species. The COI279 primer pair was suitable for samples with better DNA quality. The COI157a and COI157b primer pairs performed well for noninvasive samples and reduced the interference of nuclear mitochondrial pseudogenes (numts). COI157a could effectively identify samples from Felidae, Canidae, Viverridae, and Hyaenidae, while COI157b could be applied to samples from Ursidae, Ailuridae, Mustelidae, Procyonidae, and Herpestidae. These short‐target primers will facilitate noninvasive biological studies and efforts to conserve Carnivora species.

Carnivora, as the fourth largest order of Mammalia, harbors many top predators and threatened species (Yu & Zhang, 2006), which receive wide public attention and conservation efforts worldwide. Due to the low population size, high vigilance and elusiveness, and often nocturnal habit of these species, it is difficult to directly observe wild carnivores, which greatly hinders field surveys and conservation biology research. Under these circumstances, noninvasive sampling has become an appropriate and widely used solution for the study and conservation of wild carnivores (Zemanova, 2021), especially Felidae, Canidae, Ursidae, and Musteloidea (e.g., Bischof et al., 2016;Modi et al., 2018;Mumma et al., 2015;Wang et al., 2016).
The application of universal primers developed for vertebrates or mammals in Carnivora could pose some potential problems. First, these primers are often tested in a limited number of species, which may limit their utility in some particular groups. Second, interference from nuclear mitochondrial pseudogenes (numts) exists in some groups of Carnivora. For example, numts of COI have been reported in Panthera (Kim et al., 2006), Felis (Lopez et al., 1994), and Otocolobus (Pietsch, 2012). These universal primers might amplify numts, which would introduce errors in species identification and phylogenetic analysis (Zhang & Hewitt, 1996). Third, because DNA that is extracted from noninvasive samples is often degraded, some universal primers with relatively long product lengths might fail to amplify efficiently (Taberlet et al., 1999). Therefore, it is necessary and practical to design universal primers not only for identifying Carnivora species but also for noninvasive samples.
In this study, we designed three pairs of universal Carnivora primers using the consensus-degenerate hybrid oligonucleotide primer (CODEHOP) method (Boyce et al., 2009;Rose et al., 1998Rose et al., , 2003 and manual alignment methods and evaluated the performance of these primers using Carnivora samples from 38 species and three subspecies, which consisted of 33 tissue samples from 31 species and 20 scat samples from 19 species.

| Primer design
The CODEHOP strategy can be used to design universal primers to perform PCR amplification with distantly related gene sequences.
Designed from conserved amino acids within aligned protein sequences, a CODEHOP primer consists of a 3′ degenerate core region and a 5′ nondegenerate 'clamp' region. The 3′ core region, with a length of approximately three to four amino acids, is in a highly conserved area. To amplify more potential sequences, the 3′ core region uses a degenerate base in the third position of each codon. The 5′ clamp region, which has a length of approximately five to seven amino acids, uses the most common nucleotide of reference sequences. It does not contain degenerate bases, which ensures amplification stability and efficiency during the annealing process of the later cycles (Boyce et al., 2009;Rose et al., 1998Rose et al., , 2003 Reference COI sequences and codon usage bias were imported to Base-By-Base (version 3; Tu et al., 2018), a software package including the j-CODEHOP plug-in. The max degeneracy parameter was set to 32 to find as many potential universal Carnivora primers as possible, while clamp length was set to 15 bp and core length to four amino acids to gain primers of suitable length. Then, selected primers were evaluated by the online calculator OligoCalc (2021-3-14; Kibbe, 2007). In this step, primers were matched according to melting temperature. The poly-G/C sequence, potential hairpin formation, self-annealing, and cross-dimer were checked to filter out improper primers. A universal Carnivora primer pair COI279 (F: 5′-ATAATGATAGGAGGAttyggnaaytg; R: 5′-CATTGCAGGAGGTTTc atrttdatdatdat) was selected, in which the uppercase section is for the clamp region and the lowercase section is for the core region.
The product length was 279 bp (Figure 1), a length that could be amplified in noninvasive samples (e.g., Huber et al., 2002).
Because numts are present in Felidae as reported previously (Kim et al., 2006;Lopez et al., 1994;Pietsch, 2012), we manually designed two shorter universal Carnivora primer pairs to avoid the impacts of numts. Based on the sequence alignment results, we selected the sequences with high conservativeness among felids but multiple variations when comparing with numts, as the potential primers. Then, we used Primer Premier 5 (version 5.00; Lalitha, 2000) to assess the quality of the primer pairs based on the occurrence of hairpins, self-annealing, and cross dimers.  (Figure 1), which was a suitable length for amplifying seriously degraded noninvasive samples (Michalski et al., 2011;Santini et al., 2007). For COI279, the amplification procedure consisted of an initial denaturation (15 min at 95°C) followed by touchdown PCR (10 cycles of 30 s at a denaturation temperature of 94°C; 40 s at an annealing temperature of 62-54°C, which was decreased by 2°C every 2 cycles; and 50 s at an extension temperature of 72°C), followed by 30-35 cycles (depending on the DNA quality) of 30 s at 94°C, 40 s at 52°C, and 50 s at 72°C, and a final extension for 10 min at 72°C.

| PCR amplification
For COI157a and COI157b, the amplification procedure consisted of an initial denaturation (15 min at 95°C) followed by touchdown PCR (a total of eight cycles of 30 s at 94°C; 40 s at a temperature from 54 to 52°C, which was decreased by 2°C every four cycles; and 50 s at 72°C), followed by 30-35 cycles of 30 s at 94°C, 40 s at 50°C, and 50 s at 72°C, and a final extension for 10 min at 72°C. All PCR products were visualized in 2% agarose gels. Then, the positive products were recovered separately by Gel DNA Recovery Cleaning Kit (GeneOn Biotech) and cleaned by BigDye Sequencing Clean Up Kit (MCLAB). Finally, the products were sequenced bidirectionally on an ABI 3730XL Analyzer (Thermo Fisher, at SinoGenoMax company) to obtain reliable results.
Only clear sequence peaks were considered credible. Next, sequence searches were executed by megaBLAST of NCBI to identify the species source of the samples, and the results were then compared with the known species source of the samples.

| RE SULTS
All three pairs of universal Carnivora primers worked effectively for the Carnivora species samples. The COI279 primer pair performed well for the samples of 26 species from nine families and 23 genera of Carnivora (Table 1) Ailuropoda melanoleuca Giant panda COI279 COI157b Note: Regular without bold represents that tissue samples were successfully identified; bold means that scat samples were successfully identified; bold with underline means that both tissue and scat samples were successfully identified; N/A means that the sample was missing or exhausted; "Failed" represents the failures of PCR amplification, Sanger sequencing, or species identification. samples, the COI157b primer pair successfully identified scat samples from four of five species tested from three families (Table A4).

| DISCUSS ION
In this study, the universal Carnivora primers COI157a and COI157b performed well for most of the tested species, which represented multiple different families, and for the amplification of noninvasive samples. For most Carnivora species, species identification can be performed by combining two pairs of universal Carnivora primers.
Compared to the other primers, COI279 showed higher universality for many Carnivora species, although we lacked enough samples to test whether COI279 is suitable for pinnipeds.
Nevertheless, COI279 had some limitations. First, because the product length was 279 bp, COI279 was a better option for goodquality DNA samples. The second limitation is interference from numts. In addition to being unable to identify Panthera species, COI279 also amplified unknown sequences from Vulpes species.
Because the unknown sequences BLASTed to the Vulpes vulpes genome, we speculate that it may be the newly discovered numt of the Vulpes. This is possible because a nearly complete mitochondrial genome was discovered in the nuclear genome of dogs (Canis lupus familiaris) (Verscheure et al., 2015), implying the possible existence of unknown numts in other Canidae species.
There is no doubt that numt interference on universal primers is a large obstacle for the popularization and application of the DNA barcoding method. Coamplification could occur easily.
However, for ancient and conserved numts relative to homologous mtDNA, universal primers may tend to amplify numts rather than mtDNA (Bensasson et al., 2001). For taxonomical groups with known numts, numts could be avoided by manually designing primers. For taxonomical groups in which the existence of numts is unknown, caution is needed when analyzing the mtDNA data and especially when assembling the mitochondrial genome as a reference for DNA barcoding. Alternative methods of reducing numt interference include extracting mitochondrial DNA to reduce the impacts of numts (Ibarguchi et al., 2006) and recognizing numts by checking for the occurrence of indels, in-frame stop codons, and changes in nucleotide composition in the sequences (Song et al., 2008). Recent studies have suggested that bioinformatics filtering of high-throughput sequencing data may be a good choice for discovering unknown numts (Smart et al., 2019;Woerner et al., 2020).
For noninvasive samples, we designed COI157a and COI157b universal Carnivora primers with short PCR products, which were also named DNA minibarcoding (Meusnier et al., 2008). In our study, the universal Carnivora primers COI157a and COI157b performed well for noninvasive samples. With the rapid development of nextgeneration sequencing technology, the two primer pairs could be used as potential molecular markers for high-throughput species identification. However, in our study, short products had difficulties in differentiating subspecies or most closely related species, that is,  (Schrader et al., 2012).
In addition, it has been reported that scat samples may coamplify both predator DNA and prey DNA (Symondson, 2002). We did not find the signature of prey DNA sequences in our BLAST identification; however, this remains a problem that should be treated with caution in scat DNA studies.
Our universal Carnivora primers for species identification have some advantages over existing similar primers. For COI primers, LCO1490/HCO2198 (Folmer et al., 1994) is a widely used primer pair in Carnivora species identification. However, its 710-bp product is not suitable for noninvasive samples. Compared with our primers, the BC-F2/BC-F3/BC-R2 primer (Chaves et al., 2012), which is for Carnivora species identification, has a longer product and was tested on fewer families and species of Carnivora.
Additionally, compared with other non-COI Carnivora noninvasive primers (De Barba et al., 2014;Michalski et al., 2011;Rodriguez-Castro et al., 2020), our primers also have some advantages: for example, using only one pair of primers, obtaining DNA sequences rather than the fragment length, or having wider universality to Carnivora species.
In summary, our developed short-fragment primers could have Natural Reserve for assistance with sample collection.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflicts of interest.